Tet-On3G系统调控人巨细胞病毒蛋白pUL23稳定表达的人胚肺成纤维细胞(HELF)系的建立

Establishment of a Human Embryonic Lung Fibroblast Line Expressing Human Cytomegalovirus Tegument Protein pUL23 Regulated by Tet-On3G

人巨细胞病毒(Human cytomegalovirus,HCMV)为疱疹病毒家族一员,易导致免疫力低下或缺陷的人群严重疾病,目前对HCMV编码的皮层蛋白pUL23功能的相关报道很少。本研究应用Tet-On3G诱导表达系统,在人胚肺成纤维细胞(Human embryonic lung fibroblast,HELF)建立诱导表达HCMV病毒蛋白pUL23细胞模型。将病毒基因UL23和阳性对照基因EGFP分别定向插入应答慢病毒载体pLVX-TRE3G中,获得pLVX-TRE3GUL23-3×Flag、pLVX-TRE3G-EGFP重组慢病毒载体。运用二代慢病毒包装系统与Lenti-X293T包装细胞系,制备收获慢病毒后感染人胚肺成纤维细胞HELF。遗传霉素(Geneticin,G418)和嘌呤霉素(Puromycin,Puro)抗性筛选后多西环素(Doxycycline,Dox)诱导外源基因表达。感染后的细胞在96孔板有限稀释法成单克隆,分别采用RT-PCR与Western blot技术检测病毒UL23基因在mRNA水平与蛋白水平的表达量,筛选出当诱导时高效表达且未诱导时低表达的单克隆细胞;并评估Dox诱导剂量与诱导时间对外源蛋白pUL23表达的影响。限制性内切酶与测序显示重组质粒pLVX-TRE3G-UL23-3×Flag、pLVX-TRE3G-EGFP序列和方向正确。病毒蛋白pUL23在感染细胞中能够被诱导表达;当Dox浓度高于400ng/mL时pUL23蛋白表达量不再随Dox剂量增高而增高。在Dox诱导2h后,RT-PCR结果表明在921bp处有特定条带(UL23-3×Flag基因)与此同时,Western blot实验也检测到pUL23蛋白。这些结果表明,成功构建重组慢病毒载体,包装成慢病毒感染后,病毒基因UL23能够在人胚肺成纤维细胞中诱导表达。Dox的最佳工作浓度为400ng/mL,最佳诱导时间为12h至24h之间。这将为进一步研究病毒蛋白pU23的功能奠定基础。

The human cytomegalovirus （HCMV） is a subfamily of the betaherpesvirus. It causes significant disease in immunologically compromised individuals. The function of pUL23, a tegument protein encoded by the HCMV, is poorly understood. We applied a Lenti-X Tet-On 3G system to establish a flexible model in human embryonic lung fibroblasts （HELFs） in which pUL23 expression was induced. The UL23 gene of the HCMV and EGFP gene were obtained by a polymerase chain reaction （PCR）. The UL23 gene and EGFP gene were inserted into the response lentiviral vector pLVX-TRt^3G, respectively, to obtain the re- combinant vector pLVX-TRE3G-UL23-3 ）〈 Flag and pLVX-TRE3G-EGFP. Lentiviral supernatants were generated through a second-generation packaging system using Lenti-X293T, which was used to infect HELFs. These transduction cells screened by geneticin （G418）, and puromycin （Puro） was induced by doxycycline （Dox）. Transduction cells were seeded into 96-well plates to select monoclonal cells. Western blotting and RT-PCR were used to detect expression of the UL23 gene to seek cells with high expression efficiency and low background. In addition, the effect of pUL23 expression due to concentration and time of Dox induction were evaluated. The recombinant plasmids pLVX-TRE3G-UL23-3 ）〈 Flag and pLVX- TRE3G-EGFP were identified by DNA sequencing and restriction-enzyme digestion. In HELFs, the Dox concentration was ~400 ng/mL, so pUL23 expression was stable. Two hours after DOX induction, RT- PCR results showed that a 921-bp-specific band （UL23-3 X Flag gene） was detectable, whereas pUL23 was identified by western blotting. These results suggest that a HELF cell line stably expressing pUL23 was established with a Tet-on lentivirus expression system. The optimal concentration and time of induction was 400 ng/mL and 12--24 h, respectively. The established pUL23HELF cell line could be used as a cell model to explore the new functions of viral protein pUL23.